ELISA for VEGF

ABSTRACT

The vascular endothelial growth factor (VEGF) activity in a patient&#39;s bloodstream or other biological sample can serve as a diagnostic and prognostic index for cancer, diabetes, heart conditions, and other pathologies. Antibody-sandwich ELISA method and kits for VEGF as an antigen were developed to detect VEGF levels in biological samples from animal models and human patients and are used as a diagnostic/prognostic index.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.10/211,282, filed Aug. 5, 2002, now U.S. Pat. No. 6,885,508, which is acontinuation of U.S. application Ser. No. 09/713,515, filed Nov. 15,2000, now abandoned, which claims the benefit under 35 U.S.C. § 119(e)of U.S. Provisional Application No. 60/165,736, filed Nov. 16, 1999, theentire disclosures of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to immunoassays for detecting VEGF that can beused as diagnostic and prognostic methods for patients with cancer,cardiovascular, or other pathologies.

2. Description of Related Art

It is now well established that angiogenesis is implicated in thepathogenesis of a variety of disorders. These include solid tumors,intra-ocular neovascular syndromes such as proliferative retinopathiesor age-related macular degeneration (AMD), rheumatoid arthritis, andpsoriasis (Folkman et al. J. Biol. Chem. 267:10931-10934 (1992);Klagsbrun et al. Annu. Rev. Physiol. 53:217-239 (1991); and Garner A,Vascular diseases. In: Pathobiology of ocular disease. A dynamicapproach. Garner A, Klintworth G K, Eds. 2nd Edition (Marcel Dekker, NY,1994), pp 1625-1710). In the case of solid tumors, theneovascularization allows the tumor cells to acquire a growth advantageand proliferative autonomy compared to the normal cells. Accordingly, acorrelation has been observed between density of microvessels in tumorsections and patient survival in breast cancer as well as in severalother tumors (Weidner et al. N Engl J Med 324:1-6 (1991); Horak et al.Lancet 340:1120-1124 (1992); and Macchiarini et al. Lancet 340:145-146(1992)).

The search for positive regulators of angiogenesis has yielded manycandidates, including aFGF, bFGF, TGF-α, TGF-β, HGF, TNF-α, angiogenin,IL-8, etc. (Folkman et al., supra, and Klagsbrun et al., supra). Thenegative regulators so far identified include thrombospondin (Good etal. Proc. Natl. Acad. Sci. USA. 87:6624-6628 (1990)), the 16-kilodaltonN-terminal fragment of prolactin (Clapp et al. Endocrinology,133:1292-1299 (1993)), angiostatin (O'Reilly et al. Cell 79:315-328(1994)), and endostatin (O'Reilly et al. Cell 88:277-285 (1996)).

Work done over the last several years has established the key role ofvascular endothelial growth factor (VEGF) in the regulation of normaland abnormal angiogenesis (Ferrara et al. Endocr. Rev. 18:4-25 (1997)).The finding that the loss of even a single VEGF allele results inembryonic lethality points to an irreplaceable role played by thisfactor in the development and differentiation of the vascular system(Ferrara et al., supra).

Furthermore, VEGF has been shown to be a key mediator ofneovascularization associated with tumors and intra-ocular disorders(Ferrara et al., supra). The VEGF mRNA is overexpressed by the majorityof human tumors examined (Berkman et al. J Clin Invest 91:153-159(1993); Brown et al. Human Pathol. 26:86-91 (1995); Brown et al. CancerRes. 53:4727-4735 (1993); Mattem et al. Brit. J. Cancer. 73:931-934(1996); and Dvorak et al. Am J. Pathol. 146:1029-1039 (1995)). Also, theconcentration of VEGF in eye fluids is highly correlated to the presenceof active proliferation of blood vessels in patients with diabetic andother ischemia-related retinopathies (Aiello et al. N. Engl. J. Med.331:1480-1487 (1994)). Furthermore, studies have demonstrated thelocalization of VEGF in choroidal neovascular membranes in patientsaffected by acute macular degeneration (AMD) (Lopez et al. Invest.Ophtalmo. Vis. Sci. 37:855-868 (1996)).

VEGF is a heparin binding growth factor with a molecular weight of 45 kD(Plouet et al. EMBO J. 8:3801 (1989); Neufeld et al. Prog. Growth FactorRes. 5:89 (1994)). It is a dimeric glycoprotein consisting of twoidentical subunits. Although VEGF is encoded from a single gene, atleast five isoforms exist in vivo due to alternative mRNA splicing.These isoforms, VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206, contain121, 145, 165, 189, and 206 amino acids, respectively (Leung et al.Science 246:1306 (1989); Houck et al. Mol. Endocrinol. 5:1806 (1991);Tischer et al. J. Biol. Chem. 266:11947 (1991); Neufeld et al. The FASEBJournal 13:9-22 (1999)). The VEGF isoforms show differing affinities forheparin; VEGF121 binds heparin weakly, while VEGF165, VEGF189, andVEGF206 bind heparin with increasing affinity. VEGF121 and VEGF165 aresecreted and both isoforms are found in the circulation. In contrast,VEGF189 and VEGF206 are found mostly associated with heparin sulfatecontaining proteoglycans in the extracellular matrix (Houck et al. J.Biol. Chem. 267:26031 (1992); Park et al. Mol. Biol. Cell 4:1317(1993)). Of the five isoforms, VEGF165 is the most abundantly expressedvariant in the majority of cells and tissues.

Five receptors for VEGF have been identified: VEGFR-1 (FLT-1), VEGFR-2(KDR/FLK-1), and VEGFR-3, which are all signaling tyrosine kinases, andNeuropilin-1 and Neuropilin-2, which are both accessory-isoform-specificreceptors that bind selectively to VEGF165 (de Vries et al. Science255:989 (1992); Terman et al. Biochem. Biophys. Res. Commun. 187:1579(1992); Millauer et al. Cell 72:835 (1993); Neufeld et al., supra). Thevarious roles of these receptors in VEGF biology are under activeinvestigation by numerous groups.

VEGF is produced by tissues and does not have to enter the circulationto exert its biological effect, but rather acts locally as a paracrineregulator. This raises the question of the significance of circulatingVEGF and what role it plays in normal biology or pathology. A recentstudy by Yang et al. J. Pharm. Exp. Ther. 284:103 (1998) found theclearance of rhVEGF165 from the circulation to be very rapid, suggestingendogenous VEGF in the circulation is most likely the result ofcontinual synthesis of VEGF. In addition, several studies have tried tocorrelate levels of circulating VEGF with tumor burden and havesuggested VEGF levels as a potential prognostic marker (Ferrari andScagliotti Eur. J. Cancer 32A:2368 (1996); Gasparini et al. J. Natl.Cancer Inst. 89:139 (1997); Kohn Cancer 80:2219 (1997); Baccala et al.Urology 51:327 (1998); Fujisaki et al. Am. J. Gastroenterol. 93:249(1998)). Clearly the ability to accurately measure VEGF will beimportant to understand its potential role(s) in many biologicalprocesses, such as maintenance of vascular patency, menstrual cycle,ischemia, diabetes, and cancer.

The literature reports widely varying concentrations of endogenous VEGFin normal and diseased patients, ranging from undetectable to highlevels. It has been reported that VEGF 165/165 can be proteolyticallycleaved into three other forms: a 165/110 heterodimer, a 110/110homodimer, and a 55-amino-acid C-terminal fragment (Keyt et al. J. Biol.Chem. 271:7788-7795 (1996); Keck et al. Arch. Biochem. Biophys.344:103-113 (1997)).

The ability to measure endogenous VEGF levels depends on theavailability of sensitive and specific assays. Colorimetric,chemiluminescence, and fluorometric based enzyme-linked immunosorbentassays (ELISAs) for VEGF have been reported. Houck et al., supra,(1992); Yeo et al. Clin. Chem. 38:71 (1992); Kondo et al. Biochim.Biophys. Acta 1221:211 (1994); Baker et al. Obstet. Gynecol. 86:815(1995); Hanatani et al. Biosci. Biotechnol. Biochem. 59:1958 (1995);Leith and Michelson Cell Prolif. 28:415 (1995); Shifren et al. J. Clin.Endocrinol. Metab. 81:3112 (1996); Takano et al. Cancer Res. 56:2185(1996); Toi et al. Cancer 77:1101 (1996); Brekken et al. Cancer Res.58:1952 (1998); Obermair et al. Br. J. Cancer 77:1870-1874 (1998); Webbet al. Clin. Sci. 94:395-404 (1998). Similar ELISAs have beensuccessfully applied in the determination of low amounts of drugs andother antigenic components in plasma and urine samples, involve noextraction steps, and are simple to carry out.

The Houck et al., supra (1992) describe a colorimetric ELISA thatappears to have ng/ml sensitivity, clearly not sensitive enough todetect endogenous VEGF levels. Yeo et al., supra (1992) describe atwo-site time-resolved immunofluorometric assay, however, no VEGF wasdetected in normal sera (Yeo et al. Cancer Res. 53:2912 (1993)). Bakeret al., supra (1995), using a modified version of thisimmunofluorometric assay, reported detectable levels of VEGF in plasmafrom pregnant women, with higher levels observed in women withpreeclampsia. Similar data in pregnant women were reported by Anthony etal. Ann. Clin. Biochem. 34:276 (1997) using a radioimmunoassay. Hanataniet al., supra (1995) developed a chemiluminescent ELISA capable ofmeasuring circulating VEGF and report VEGF levels in sera from 30 normalindividuals (male and female) from 8-36 pg/ml. Brekken et al., supra(1998) described ELISA assays using antibodies having binding preferenceto either the VEGF alone or the VEGF:Flk-1 complex.

An ELISA kit for VEGF detection is commercially available from R&DSystems (Abingdon, U.K.). The R&D VEGF ELISA kit has been used insandwich assays wherein a monoclonal antibody is used to capture thetarget VEGF antigen and a polyclonal antibody is used to detect theVEGF. Webb et al. supra (1998). It is not clear whether the detectionresults using the R&D ELISA kit are influenced by the presence ofproteolytical processes or degradation of VEGF, or by interference ofother serum proteins. Obermair et al., supra (1998).

Keyt et al. J. Biol. Chem. 271:7788-7795 (1996); Keyt et al. J. Biol.Chem. 271:5638 (1996); and Shifren et al., supra (1996) also developed acolorimetric ELISA based on a dual monoclonal antibody pair. Althoughthis ELISA was able to detect elevated VEGF levels in cancer patients,it lacked the sensitivity needed to measure endogenous levels of VEGF innormal individuals. Rodriguez et al. J. Immunol. Methods 219:45 (1998)described a two-site fluorimetric VEGF ELISA that yields a sensitivityof 10 pg/ml VEGF in neat plasma or serum. However, this fluorimetricassay can only detect fully intact 165/165 and 165/110 species of VEGF.

Thus, there is a need to develop a diagnostic and prognostic assay thatdetects higher measurable levels of VEGF in a biological sample of ananimal model or patient than existing ELISAs, and can measure all theisoforms of VEGF.

SUMMARY OF THE INVENTION

A multi-site antibody-sandwich ELISA method and kits for VEGF as antigenwere developed to detect VEGF in biological samples and used as adiagnostic/prognostic index. Compared to the currently used VEGF ELISAs,the present assay has high sensitivity and is capable of detecting mostof the isoforms of endogenous VEGF in circulation.

Specifically, the invention provides a method for detecting VEGF in abiological sample, preferably from vascular, diabetic, or cancerpatients, comprising the steps of:

(a) contacting and incubating the biological sample with pre-mixedcapture reagents immobilized to a solid support, wherein the capturereagents are polyclonal and monoclonal antibodies against human VEGF,said monoclonal antibody binding specifically to the C-terminal(residues 111-165) of human VEGF;

(b) separating the biological sample from the immobilized capturereagents;

(c) contacting the immobilized capture reagents with a detectableantibody that binds to the KDR and FLT1 receptor binding domains ofVEGF; and

(d) measuring the level of VEGF bound to the capture reagents using adetection means for the detectable antibody.

Preferably, the capture reagents are immobilized in a weight ratio ofabout 0.8:1 to 1.2:1 of monoclonal to polyclonal antibody. Morepreferably, the weight ratio is about 1:1 of monoclonal to polyclonalantibody.

In another aspect, the invention provides an immunoassay kit fordetecting VEGF in a biological sample, the kit comprising:

(a) as capture reagents, polyclonal and monoclonal antibodies againsthuman VEGF premixed in a weight ratio of about 0.8:1 to 1.2:1 ofmonoclonal to polyclonal antibody, wherein the monoclonal antibody bindsspecifically to the C-terminal (residues 111-165) of human VEGF; and

(b) as detection reagent, a detectable antibody that binds to the KDRand FLT1 receptor binding domains of VEGF.

The assay herein is unique in that it uses a polyclonal/monoclonalantibody mixture as the capture reagents, and the capture monoclonalantibody binds to the C-terminal portion of VEGF. Most of the previouslydisclosed VEGF ELISAs are based on either a dual monoclonal antibodypair for capture/detection, or a monoclonal antibody as capture reagentand a polyclonal antibody for detection. If a polyclonal antibody isused alone as the capture antibody, all sensitivity of the assay islost. The ability of the monoclonal capture antibody to bind the VEGFC-terminus ensures that all the endogenous VEGF molecules, including165/165, 165/110 and 110/110 can be detected by the assay describedherein. Furthermore, the detection antibody of the invention binds tothe biologically active regions of VEGF, i.e., the binding domains forthe KDR and FLT1 receptors of VEGF, which ensures that the detected VEGFmolecules are free from being blocked by, for example, soluble VEGFreceptors in the circulation. As such, the assay described hereinprovides a more accurate measurement of circulating VEGF molecules thatare most likely biologically active.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparison of two different preparations ofaffinity-purified rabbit polyclonal antibody against rhVEGF, with thesquares depicting the preferred antibody and the circles representingthe same antibody from a different bleed.

FIG. 2 shows a comparison of ELISAs herein using guanidine versusglycine elution of the affinity-purified rabbit polyclonal antibodyagainst VEGF.

FIG. 3 shows a comparison of typical standard curves for, and possiblehook effect of, three different VEGF ELISAs, wherein the circles showthe one-site assay with MAb 3.5F8 alone as coat and detection agent, thesquares show the two-site assay with MAb 3.5F8 as coat and MAb A4.6.1 asdetection agent, and the diamonds show the multi-site assay herein withMAb 3.5F8 and an affinity-purified polyclonal antibody as coat and MAbA4.6.1 as detection agent.

FIG. 4 shows monoclonal antibody MAb 3.5F8 coat maximization wherein theELISA uses 0.4 (circles), 1 (squares), 2 (diamonds), or 4 (triangles)μg/ml monoclonal antibody and 1 μg/ml affinity-purified polyclonalantibody.

FIG. 5 shows rabbit polyclonal antibody coat maximization wherein theELISA uses 0 (circles), 0.1 (squares), 0.4 (diamonds), 1 (triangles), 2(reverse triangles with dotted lines), or 4 (reverse triangles withsolid lines) μg/ml affinity-purified polyclonal antibody and 0.4 μg/mlMAb 3.5F8.

FIG. 6 shows the effect of pH on the multi-site VEGF ELISA herein,wherein the circles represent the ELISA at pH 4, the squares, pH 5, thediamonds, pH 6, the triangles, pH 7, the half-line diamonds, pH 8, andthe reverse diamonds, pH 9.

FIG. 7 shows dilution linearity of six normal human EDTA plasma samplesspiked with rhVEGF in the multi-site VEGF ELISA.

FIGS. 8A-8C show linearity of normal rat EDTA plasma samples spiked withrhVEGF, wherein FIG. 8A shows high spike, FIG. 8B shows mid-spike, andFIG. 8C shows low spike, and wherein the circles are rat pool 1, thesquares are rat pool 2, and the diamonds are rat 1.

FIGS. 9A-9B show linearity of, respectively, four female and four maleYorkshire pig EDTA plasma samples spiked with rhVEGF.

FIGS. 10A-10C show the specificity of three different ELISA assays forVEGF forms 165/165 (circles), 165/110 (squares), 121/121 (diamonds), and110/110 (triangles). FIG. 10A shows the specificity of the single-siteELISA using MAb 3.5F8 as coat and detection antibody (FIG. 10A), FIG.10B shows the specificity of the two-site fluorimetric VEGF ELISA assayusing MAb 3.5F8 as coat and MAb A4.6.1 as detection antibody, and FIG.10C shows the specificity of the multi-site VEGF ELISA herein using MAb3.5F8 and PAb as coat antibodies and MAb A4.6.1 as detection antibody.

FIGS. 11A and 11B show, respectively, normal human plasma and serum VEGFdetected by a two-site ELISA using only MAb 3.5F8 as coat reagent and bythe multi-site ELISA herein using both the PAb and MAb 3.5F8 as coatreagents.

FIG. 12 shows the amounts of plasma VEGF in cardiac patients using allthree assays described in the legend to FIG. 10, where the circlesrepresent the single-site assay, the squares represent the two-siteassay, and the triangles represent the multi-site assay.

FIG. 13 shows plasma VEGF levels in normal donors and cardiovascularpatients using the two-site assay with MAb 3.5F8 as coat and MAb A4.6.1as detection antibody or the multi-site assay herein using MAb 3.5F8 andaffinity-purified polyclonal antibody as coat and MAb A4.6.1 asdetection antibody, where N is the number of patients.

FIG. 14 shows serum VEGF from lung cancer patients detected by atwo-site ELISA using only MAb 3.5F8 as coat reagent and by themulti-site ELISA herein using both the PAb and MAb 3.5F8 as coatreagents.

FIG. 15 shows serum VEGF levels in normal donors and diabetic patients(non-insulin-dependent diabetes mellitus (NIDDM) and insulin-dependentdiabetes mellitus (IDDM)) using the two-site ELISA with MAb 3.5F8 ascoat and MAb A4.6.1 as detection antibody.

FIGS. 16A and 16B show graphs comparing an affinity-purified polyclonalantibody to DNase and an affinity-purified polyclonal antibody to VEGFas one of the coat reagents in the multi-site assay herein, as comparedto the two-site assay using MAb 3.5F8 as coat reagent for human plasma.FIG. 16A shows correlation of plasma VEGF measured by the two-site ELISAusing MAb 3.5F8 as the coat reagent (x-axis) with plasma VEGF measuredby a multi-site assay with MAb 3.5F8 and a polyclonal antibody to DNaseas the coat reagents (filled circles), and with plasma VEGF measured bya multi-site assay as set forth herein using MAb 3.5F8 and the PAb toVEGF as the coat reagents (open circles). FIG. 16B shows the standardcurves of the two-site ELISA (filled circles), the multi-site ELISA withMAb 3.5F8 and a polyclonal antibody to DNase as the coat reagents(filled diamonds), and a multi-site assay as set forth herein using MAb3.5F8 and the PAb to VEGF as the coat reagents (filled squares).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A. Definitions

The term “VEGF” as used herein refers to the 165-amino acid vascularendothelial cell growth factor, and related 121-, 145-, 189-, and206-amino acid vascular endothelial cell growth factors, as described byLeung et al. Science 246:1306 (1989), Houck et al. Mol. Endocrin. 5:1806(1991), and Neufeld et al., supra, together with the naturally occurringallelic and processed forms of those growth factors.

The term “detecting” is used in the broadest sense to include bothqualitative and quantitative measurements of a target molecule. In oneaspect, the detecting method as described herein is used to identify themere presence of VEGF in a biological sample. In another aspect, themethod is used to test whether VEGF in a sample is at a detectablelevel. In yet another aspect, the method can be used to quantify theamount of VEGF in a sample and further to compare the VEGF levels fromdifferent samples.

The term “biological sample” refers to a body sample from any animal,but preferably is from a mammal, more preferably from a human. Mostpreferably, such biological sample is from vascular, diabetic, or cancerpatients. Such samples include biological fluids such as serum, plasma,vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminalfluid, amniotic fluid, milk, whole blood, urine, cerebro-spinal fluid,saliva, sputum, tears, perspiration, mucus, and tissue culture medium,as well as tissue extracts such as homogenized tissue, and cellularextracts. The preferred biological sample herein is serum, plasma orurine.

The term “capture reagent” refers to a reagent capable of binding andcapturing a target molecule in a sample such that under suitablecondition, the capture reagent-target molecule complex can be separatedfrom the rest of the sample. Typically, the capture reagent isimmobilized or immobilizable. In a sandwich immunoassay, the capturereagent is preferably an antibody or a mixture of different antibodiesagainst a target antigen.

The term “detectable antibody” refers to an antibody that is capable ofbeing detected either directly through a label amplified by a detectionmeans, or indirectly through, e.g., another antibody that is labeled.For direct labeling, the antibody is typically conjugated to a moietythat is detectable by some means. The preferred detectable antibody isbiotinylated antibody.

The term “detection means” refers to a moiety or technique used todetect the presence of the detectable antibody in the ELISA herein andincludes detection agents that amplify the immobilized label such aslabel captured onto a microtiter plate. Preferably, the detection meansis a fluorimetric detection agent such as avidin or streptavidin.

The term “antibody” is used in the broadest sense and includesmonoclonal antibodies (including agonist, antagonist, and neutralizingantibodies), polyclonal antibodies, multivalent antibodies,multispecific antibodies, and antibody fragments so long as they exhibitthe desired binding specificity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally-occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations that typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al. Nature256:495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al. Nature 352:624-628 (1991) and Marks et al. J. Mol. Biol.222:581-597 (1991), for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al. Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which hypervariable regionresidues of the recipient are replaced by hypervariable region residuesfrom a non-human species (donor antibody) such as mouse, rat, rabbit ornonhuman primate having the desired specificity, affinity, and capacity.In some instances, framework region (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable regions correspond to those of anon-human immunoglobulin and all or substantially all of the FRs arethose of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al. Nature 321:522-525 (1986); Reichmannet al. Nature 332:323-329 (1988); and Presta Curr. Op. Struct. Biol.2:593-596 (1992).

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic, and farm animals, and zoo, sports,or pet animals, such as dogs, horses, cats, sheep, pigs, cows, etc.Preferably, the mammal is human.

The terms “cancer”, “cancerous”, and “malignant” refer to or describethe physiological condition in mammals that is typically characterizedby unregulated cell growth. Examples of cancer include but are notlimited to, carcinoma including adenocarcinoma, lymphoma, blastoma,melanoma, sarcoma, and leukemia. More particular examples of suchcancers include squamous cell cancer, small-cell lung cancer, non-smallcell lung cancer, gastrointestinal cancer, Hodgkin's and non-Hodgkin'slymphoma, pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, liver cancer such as hepatic carcinoma and hepatoma, bladdercancer, breast cancer, colon cancer, colorectal cancer, endometrialcarcinoma, salivary gland carcinoma, kidney cancer such as renal cellcarcinoma and Wilms' tumors, basal cell carcinoma, melanoma, prostatecancer, vulval cancer, thyroid cancer, testicular cancer, esophagealcancer, and various types of head and neck cancer. The preferred cancersfor treatment herein are breast, colon, lung, and melanoma.

The phrases “vascular” and “cardiovascular” are used interchangeably anddescribe patients with indications that stimulate angiogenesis and/orcardiovascularization, and those that inhibit angiogenesis and/orcardiovascularization. Such disorders include, for example, arterialdisease, such as atherosclerosis, hypertension, inflammatory vasculitis,Reynaud's disease and Reynaud's phenomenon, aneurysms, and arterialrestenosis; venous and lymphatic disorders such as thrombophlebitis,lymphangitis, and lymphedema; and other vascular disorders such asperipheral vascular disease, cancer such as vascular tumors, e.g.,hemangioma (capillary and cavernous), glomus tumors, telangiectasia,bacillary angiomatosis, hemangioendothelioma, angiosarcoma,haemangiopericytoma, Kaposi's sarcoma, lymphangioma, andlymphangiosarcoma, tumor angiogenesis, trauma such as wounds, burns, andother injured tissue, implant fixation, scarring, ischemia reperfusioninjury, rheumatoid arthritis, cerebrovascular disease, renal diseasessuch as acute renal failure, and osteoporosis. This would also includeangina, myocardial infarctions such as acute myocardial infarctions,cardiac hypertrophy, and heart failure such as congestive heart failure(CHF).

The term “diabetes” refers to a progressive disease of carbohydratemetabolism involving inadequate production or utilization of insulin andis characterized by hyperglycemia and glycosuria. This term includes allforms of diabetes, such as type I and type II diabetes andinsulin-resistant diabetes, such as Mendenhall's Syndrome, WernerSyndrome, leprechaunism, lipoatrophic diabetes, and other lipoatrophies.

The term “affinity purified” refers to purifying a substance by elutingit through an affinity chromatography column.

B. Modes for Carrying Out the Invention

The assay described herein is a multi-site immunoassay utilizing thefollowing steps.

First Step

In the first step of the assay herein, the biological sample iscontacted and incubated with the immobilized capture (or coat) reagents,which are an anti-VEGF monoclonal antibody and a polyclonal antibodydirected against VEGF. These antibodies may be from any species, butpreferably the monoclonal antibody is a murine or rat monoclonalantibody, more preferably murine, and most preferably MAb 3.5F8(Rodriguez et al., supra (1998)), and the polyclonal antibody rabbitanti-VEGF or goat anti-VEGF antibody, more preferably rabbit anti-VEGF.Furthermore, the polyclonal antibody is preferably affinity purified, todecrease background. Hence, in a specific preferred embodiment, theimmobilized monoclonal antibody is a murine monoclonal antibody, mostpreferably MAb 3.5F8, and the immobilized polyclonal antibody is anaffinity-purified rabbit antibody. The immobilized capture reagents aremixed together before they are immobilized. Immobilizationconventionally is accomplished by insolubilizing the capture reagentseither before the assay procedure, as by adsorption to a water-insolublematrix or surface (U.S. Pat. No. 3,720,760) or non-covalent or covalentcoupling (for example, using glutaraldehyde or carbodiimidecross-linking, with or without prior activation of the support with,e.g., nitric acid and a reducing agent as described in U.S. Pat. No.3,645,852 or in Rotmans et al. J. Immunol. Methods 57:87-98 (1983)), orafterward, e.g., by immunoprecipitation.

The solid phase used for immobilization may be any inert support orcarrier that is essentially water insoluble and useful in immunometricassays, including supports in the form of, e.g., surfaces, particles,porous matrices, etc. Examples of commonly used supports include smallsheets, Sephadex, polyvinyl chloride, plastic beads, and assay plates ortest tubes manufactured from polyethylene, polypropylene, polystyrene,and the like including 96-well microtiter plates, as well as particulatematerials such as filter paper, agarose, cross-linked dextran, and otherpolysaccharides. Alternatively, reactive water-insoluble matrices suchas cyanogen bromide-activated carbohydrates and the reactive substratesdescribed in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642;4,229,537; and 4,330,440 are suitably employed for capture reagentimmobilization. In a preferred embodiment the immobilized capturereagents are coated on a microtiter plate, and in particular thepreferred solid phase used is a multi-well microtiter plate that can beused to analyze several samples at one time. The most preferred is amicrotest 96-well ELISA plate such as that sold as Nune Maxisorb orImmulon.

The solid phase is coated with the pre-mixed capture reagents as definedabove, which may be linked by a non-covalent or covalent interaction orphysical linkage as desired. Techniques for attachment include thosedescribed in U.S. Pat. No. 4,376,110 and the references cited therein.If covalent, the plate or other solid phase is incubated with across-linking agent together with the capture reagent under conditionswell known in the art such as for 1 hour at room temperature.

Commonly used cross-linking agents for attaching the pre-mixed capturereagents to the solid phase substrate include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis (succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates capable of forming cross-links in the presence of light.

If 96-well plates are utilized, they are preferably coated with themixture of capture reagents (typically diluted in a buffer such as 0.05M sodium carbonate by incubation for at least about 10 hours, morepreferably at least overnight, at temperatures of about 4-20° C., morepreferably about 4-8° C., and at a pH of about 8-12, more preferablyabout 9-10, and most preferably about 9.6. If shorter coating times (1-2hours) are desired, one can use 96-well plates with nitrocellulosefilter bottoms (Millipore MULTISCREEN™) or coat at 37° C. The plates maybe stacked and coated long in advance of the assay itself, and then theassay can be carried out simultaneously on several samples in a manual,semi-automatic, or automatic fashion, such as by using robotics.

The coated plates are then typically treated with a blocking agent thatbinds non-specifically to and saturates the binding sites to preventunwanted binding of the free ligand to the excess sites on the wells ofthe plate. Examples of appropriate blocking agents for this purposeinclude, e.g., gelatin, bovine serum albumin, egg albumin, casein, andnon-fat milk. The blocking treatment typically takes place underconditions of ambient temperatures for about 1-4 hours, preferably about1.5 to 3 hours.

After coating and blocking, the VEGF standard (purified VEGF) or thebiological sample to be analyzed, appropriately diluted, is added to theimmobilized phase. The preferred dilution rate is about 5-15%,preferably about 10%, by volume. Buffers that may be used for dilutionfor this purpose include (a) PBS containing 0.5% BSA, 0.05% TWEEN 20™detergent (P20), 0.05% PROCLIN™ 300 antibiotic, 5 mM EDTA, 0.25% Chapssurfactant, 0.2% beta-gamma globulin, and 0.35M NaCl; (b) PBS containing0.5% BSA, 0.05% P20, and 0.05% PROCLIN™ 300, pH 7; (c) PBS containing0.5% BSA, 0.05% P20, 0.05% PROCLIN™ 300, 5 mM EDTA, and 0.35 M NaCl, pH6.35; (d) PBS containing 0.5% BSA, 0.05% P20, 0.05% PROCLIN™ 300, 5 mMEDTA, 0.2% beta-gamma globulin, and 0.35 M NaCl; and (e) PBS containing0.5% BSA, 0.05% P20, 0.05% PROCLIN™ 300, 5 mM EDTA, 0.25% Chaps, and0.35 M NaCl. Buffer (c) is the preferred buffer for the assay hereinsince it has the best differentiation between each standard as well asthe biggest signal-to-noise ratio. PROCLIN™ 300 acts as a preservative,and TWEEN 20™ acts as a detergent to eliminate non-specific binding. Theadded EDTA and salt of buffer (c) act to decrease the background overthe other buffers, including buffer (b).

The weight ratio of the capture reagents (monoclonal antibody topolyclonal antibody) is preferably about 0.8:1 to about 1.2:1, morepreferably about 1:1. The amount of capture reagents employed issufficiently large to give a good signal in comparison with the VEGFstandards, but not in molar excess compared to the maximum expectedendogenous VEGF level in the sample. For sufficient sensitivity, it ispreferred that the amount of biological sample added be such that theimmobilized capture reagents are in molar excess of the maximum molarconcentration of free VEGF anticipated in the biological sample afterappropriate dilution of the sample. This anticipated level dependsmainly on any known correlation between the concentration levels of thefree VEGF in the particular biological sample being analyzed with theclinical condition of the patient. Thus, for example, cancer patientsmay have a maximum expected concentration of free VEGF in their serumthat is quite high, whereas a normal child or adult will be expected tohave a much lower level of free VEGF in their serum based on what isknown in the literature.

If too much of the capture reagents are present, however, the capturereagents will compete with the anti-VEGF present in the biologicalsample for the bound VEGF, yielding inaccurate results. Thus, while theconcentration of the capture reagents will generally be determined bythe concentration range of interest of the VEGF taking any necessarydilution of the biological sample into account, the final concentrationof the capture reagents will normally be determined empirically tomaximize the sensitivity of the assay over the range of interest.However, as a general guideline, the molar excess is suitably less thanabout ten-fold of the maximum expected molar concentration of free VEGFin the biological sample after any appropriate dilution of the sample.Most preferably, the amount of monoclonal antibodies immobilized isabout 0.4 μg/ml and the amount of polyclonal antibodies immobilized isabout 0.4 μg/ml.

The conditions for incubation of sample and immobilized capture reagentare selected to maximize sensitivity of the assay and to minimizedissociation. Preferably, the incubation is accomplished at fairlyconstant temperatures, ranging from about 0° C. to about 40° C.,preferably from about 36 to 38° C. to obtain a less variable, lowercoefficient of variant (CV) than at, e.g, room temperature. The time forincubation depends primarily on the temperature, being generally nogreater than about 10 hours to avoid an insensitive assay. Preferably,the incubation time is from about 0.5 to 3 hours, and more preferably1.5-3 hours at 36-38° C. to maximize binding of free VEGF to capturereagents. The duration of incubation may be longer if a proteaseinhibitor is added to prevent proteases in the biological fluid fromdegrading the VEGF.

At this stage, the pH of the incubation mixture will ordinarily be inthe range of about 6-9.5, preferably in the range of about 6-7, morepreferably about 6.0 to 6.5, and most preferably the pH of the assay(ELISA) diluent is 6.35±0.1. Acidic pH such as pH 4-5 decreased recoveryof VEGF. The pH of the incubation buffer is chosen to maintain asignificant level of specific binding of the capture reagents to theVEGF being captured. Various buffers may be employed to achieve andmaintain the desired pH during this step, including borate, phosphate,carbonate, Tris-HCl or Tris-phosphate, acetate, barbital, and the like.The particular buffer employed is not critical to the invention, but inindividual assays one buffer may be preferred over another.

Second Step

In the second step of the assay method herein, the biological sample isseparated (preferably by washing) from the immobilized capture reagentsto remove uncaptured VEGF. The solution used for washing is generally abuffer (“washing buffer”) with a pH determined using the considerationsand buffers described above for the incubation step, with a preferablepH range of about 6-9. The washing may be done three or more times. Thetemperature of washing is generally from refrigerator to moderatetemperatures, with a constant temperature maintained during the assayperiod, typically from about 0-40° C., more preferably about 4-30° C.For example, the wash buffer can be placed in ice at 4° C. in areservoir before the washing, and a plate washer can be utilized forthis step. A cross-linking agent or other suitable agent may also beadded at this stage to allow the now-bound VEGF to be covalentlyattached to the capture reagents if there is any concern that thecaptured VEGF may dissociate to some extent in the subsequent steps.

Third Step

In the next step, the immobilized capture reagents are contacted withdetectable antibodies, preferably at a temperature of about 20-40° C.,more preferably about 36-38° C., with the exact temperature and time forcontacting the two being dependent primarily on the detection meansemployed. For example, when 4-methylumbelliferyl-β-galactoside (MUG) andstreptavidin-β-galactosidase are used as the means for detection,preferably the contacting is carried out overnight (e.g., about 15-17hours or more) to amplify the signal to the maximum. While thedetectable antibody may be a polyclonal or monoclonal antibody,preferably it is a monoclonal antibody, more preferably murine, and mostpreferably MAb A4.6.1. Also, the preferred detectable antibody isdirectly detectable, and preferably has a fluorimetric label. Thefluorimetric label has greater sensitivity to the assay compared to theconventional colorimetric label. More preferably, the detectableantibody is biotinylated and the detection means is avidin orstreptavidin-β-galactosidase and MUG.

Preferably a molar excess of an antibody with respect to the maximumconcentration of free VEGF expected (as described above) is added to theplate after it is washed. This antibody (which is directly or indirectlydetectable) is preferably a polyclonal antibody, although any antibodycan be employed. The affinity of the antibody must be sufficiently highthat small amounts of the free VEGF can be detected, but not so highthat it causes the VEGF to be pulled from the capture reagents.

Fourth Step

In the last step of the assay method, the level of free VEGF that is nowbound to the capture reagents is measured using a detection means forthe detectable antibody. If the biological sample is from a vascular,diabetic, or cancer patient, the measuring step preferably comprisescomparing the reaction that occurs as a result of the above three stepswith a standard curve to determine the level of VEGF compared to anormal individual.

Antibody Production

Polyclonal antibodies to the VEGF generally are raised in animals bymultiple subcutaneous (sc) or intraperitoneal (ip) injections of theVEGF and an adjuvant. It may be useful to conjugate the VEGF or afragment containing the target amino acid sequence to a protein that isimmunogenic in the species to be immunized, e.g., keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsininhibitor using a bifunctional or derivatizing agent, for example,maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteineresidues), N-hydroxysuccinimide (through lysine residues),glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, where R and R¹are different alkyl groups.

The antibodies used as the coat or detectable antibodies may be obtainedfrom any convenient vertebrate source, such as murine, primate,lagomorpha, goat, rabbit, rat, chicken, bovine, ovine, equine, canine,feline, or porcine. Chimeric or humanized antibodies may also beemployed, as described, e.g., in U.S. Pat. No. 4,816,567; Morrison etal. Proc. Natl. Acad. Sci. USA 81:6851 (1984); Neuberger et al. Nature312: 604 (1984); Takeda et al. Nature 314:452 (1985); and WO 98/45331published Oct. 15, 1998, as well as in those additional references setforth above.

Animals may be immunized against the immunogenic conjugates orderivatives by combining 1 mg or 1 μg of conjugate (for rabbits or mice,respectively) with 3 volumes of Freund's complete adjuvant and injectingthe solution intradermally at multiple sites. One month later theanimals are boosted with ⅕ to 1/10 the original amount of conjugate inFreund's incomplete adjuvant by subcutaneous injection at multiplesites. 7 to 14 days later animals are bled and the serum is assayed foranti-VEGF titer. Animals are boosted until the titer plateaus.Preferably, the animal is boosted with the conjugate of VEGF, butconjugated to a different protein and/or through a differentcross-linking agent. Conjugates also can be made in recombinant cellculture as protein fusions. Also, aggregating agents such as alum areused to enhance the immune response. Methods for the production ofpolyclonal antibodies are described in numerous immunology textbooks,such as Davis et al. Microbiology, 3rd Edition, (Harper & Row, New York,N.Y., 1980).

Monoclonal antibodies are prepared by recovering spleen cells fromimmunized animals and immortalizing the cells in conventional fashion,e.g. by fusion with myeloma cells or by Epstein-Barr virustransformation, and screening for clones expressing the desiredantibody. See, e.g., Kohler and Milstein Eur. J. Immunol. 6:511 (1976).Monoclonal antibodies, or the antigen-binding region of a monoclonalantibody, such as Fab or (Fab)₂ fragments, may alternatively be producedby recombinant methods.

Examples of suitable antibodies include those already utilized in knownRIAs for the protein in question, e.g., those antibodies directedagainst VEGF as described in the references given in the introductionherein.

Detection

The antibody added to the immobilized capture reagents will be eitherdirectly labeled, or detected indirectly by addition, after washing offof excess first antibody, of a molar excess of a second, labeledantibody directed against IgG of the animal species of the firstantibody. In the latter, indirect assay, labeled antisera against thefirst antibody are added to the sample so as to produce the labeledantibody in situ.

The label used for either the first or second antibody is any detectablefunctionality that does not interfere with the binding of free VEGF tothe antibody. Examples of suitable labels are those numerous labelsknown for use in immunoassay, including moieties that may be detecteddirectly, such as fluorochrome, chemiluminscent, and radioactive labels,as well as moieties, such as enzymes, that must be reacted orderivatized to be detected. Examples of such labels include theradioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I, fluorophores such as rareearth cheats or fluorescein and its derivatives, rhodamine and itsderivatives, dansyl, umbelliferone, luceriferases, e.g., fireflyluciferase and bacterial luciferase (U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme,saccharide oxidases, e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricaseand xanthine oxidase, coupled with an enzyme that employs hydrogenperoxide to oxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase, biotin/avidin, biotin/streptavidin,biotin/Streptavidin-β-galactosidase with MUG, spin labels, bacteriophagelabels, stable free radicals, and the like. As noted above, thefluorimetric detection is preferred.

Conventional methods are available to bind these labels covalently toproteins or polypeptides. For instance, coupling agents such asdialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotizedbenzidine, and the like may be used to tag the antibodies with theabove-described fluorescent, chemiluminescent, and enzyme labels. See,for example, U.S. Pat. Nos. 3,940,475 (fluorimetry) and 3,645,090(enzymes); Hunter et al. Nature 144:945 (1962); David et al.Biochemistry 13:1014-1021 (1974); Pain et al. J. Immunol. Methods40:219-230 (1981); and Nygren J. Histochem. and Cytochem. 30:407-412(1982). Preferred labels herein are fluorescent to increaseamplification and sensitivity to 8 pg/ml, more preferably biotin withstreptavidin-β-galactosidase and MUG for amplifying the signal.

The conjugation of such label, including the enzymes, to the antibody isa standard manipulative procedure for one of ordinary skill inimmunoassay techniques. See, for example, O'Sullivan et al. “Methods forthe Preparation of Enzyme-antibody Conjugates for Use in EnzymeImmunoassay,” in Methods in Enzymology, ed. J. J. Langone and H. VanVunakis, Vol. 73 (Academic Press, New York, N.Y., 1981), pp. 147-166.

Following the addition of last labeled antibody, the amount of boundantibody is determined by removing excess unbound labeled antibodythrough washing and then measuring the amount of the attached labelusing a detection method appropriate to the label, and correlating themeasured amount with the amount of free VEGF in the biological sample.For example, in the case of enzymes, the amount of color developed andmeasured will be a direct measurement of the amount of VEGF present.Specifically, if HRP is the label, the color is detected using thesubstrate OPD at 490 nm absorbance.

In one example, after an enzyme-labeled second antibody directed againstthe first unlabeled antibody is washed from the immobilized phase, coloror chemiluminiscence is developed and measured by incubating theimmobilized capture reagent with a substrate of the enzyme. Then theamount of free VEGF concentration is calculated by comparing with thecolor or chemiluminescence generated by the standard VEGF run inparallel.

Kits

As a matter of convenience, the assay method of this invention can beprovided in the form of a kit. Such a kit is a packaged combinationincluding the basic elements of:

(a) capture reagents comprised of polyclonal and monoclonal antibodiesagainst human VEGF molecule, wherein the monoclonal antibody bindsspecifically to the C-terminal of the VEGF molecule, in a weight ratioof about 0.8:1 to 1.2:1 of monoclonal to polyclonal antibody; and

(b) detection reagents comprised of detectable (labeled or unlabeled)antibodies that bind to the KDR and FLT1 receptor binding domains ofVEGF.

These basic elements are defined hereinabove.

Preferably, the kit further comprises a solid support for the capturereagents, which may be provided as a separate element or on which thecapture reagents are already immobilized. Hence, the capture antibodiesin the kit may be immobilized on a solid support, or they may beimmobilized on such support that is included with the kit or providedseparately from the kit. Preferably, the capture reagents are coated ona microtiter plate. The detection reagent may be labeled antibodiesdetected directly or unlabeled antibodies that are detected by labeledantibodies directed against the unlabeled antibodies raised in adifferent species. Where the label is an enzyme, the kit will ordinarilyinclude substrates and cofactors required by the enzyme, and where thelabel is a fluorophore, a dye precursor that provides the detectablechromophore. Where the detection reagent is unlabeled, the kit mayfurther comprise a detection means for the detectable antibodies, suchas the labeled antibodies directed to the unlabeled antibodies,preferably in a fluorimetric-detected format.

In a preferred specific embodiment, the weight ratio of monoclonalantibody to polyclonal antibody in the kit is about 1:1, the detectableantibody is a biotinylated murine monoclonal antibody, the monoclonalantibody is murine or rat, more preferably murine, and most preferablyMAb 3.5F8, the polyclonal antibody is affinity purified, and morepreferably from goat or rabbit, most preferably rabbit, and the amountof murine monoclonal antibodies is 0.4 μg/ml and the amount of rabbitpolyclonal antibodies is 0.4 μg/ml. Preferably, the capture reagents areimmobilized in this kit. Also, preferably the detectable antibody is MAbA4.6.1.

The kit also typically contains instructions for carrying out the assay,and/or VEGF as an antigen standard (e.g., purified VEGF, preferablyrecombinantly produced VEGF), as well as other additives such asstabilizers, washing and incubation buffers, and the like.

Examples of standards for VEGF are recombinant human VEGF produced inmammalian cells available from Genentech, Inc., South San Francisco,Calif.

The components of the kit will be provided in predetermined ratios, withthe relative amounts of the various reagents suitably varied to providefor concentrations in solution of the reagents that substantiallymaximize the sensitivity of the assay. Particularly, the reagents may beprovided as dry powders, usually lyophilized, including excipients,which on dissolution will provide for a reagent solution having theappropriate concentration for combining with the sample to be tested.

The following examples are intended to illustrate one embodiment nowknown for practicing the invention, but the invention is not to beconsidered limited to these examples. All open and patented literaturecitations herein are expressly incorporated by reference.

EXAMPLE 1

2. Materials and Methods

2.1. Reagents

Purified recombinant human VEGF165 (rhVEGF) expressed in Escherichiacoli (Genentech, South San Francisco, Calif.) was used as the standardand for the controls (prepared in ELISA diluent as defined below andstored at −70° C.). Streptavidin-β-galactosidase (Strep-β-gal) waspurchased from Boehringer Mannheim, W. Germany; MUG was purchased fromSigma, St. Louis, Mo. Dimethylsulfoxide (DMSO) was purchased from Sigma.

2.2. Antibodies to VEGF

Antibodies against rhVEGF165 were prepared as described in Kim et al.,Growth Factors, 7:53 (1992). Briefly, BALB/c mice were hyperimmunizedintraperitoneally with a 10 mg dose of rhVEGF165 conjugated to keyholelimpet hemocyanin. Spleen cells were fused with a mouse myeloma line andculture supernatants from wells containing hybridomas were screened forthe presence of MAbs to rhVEGF165 by an ELISA. Positive hybridomas werecloned twice using the limiting dilution technique. The monoclonalantibodies used in this ELISA have been characterized in Kim et al.,supra (1992). One of the capture antibodies, MAb 3.5F8, is thought tobind near the heparin binding domain, amino acid residues 111-165, witha K_(d) of 13 pM. Rodriguez et al., supra (1998).

The rabbit polyclonal antibody (PAb) used as the other coat antibody wasgenerated by injecting VEGF into a rabbit using a standard protocol, andpurified by passing it through an affinity column to which VEGF wascoupled to capture the polyclonal antibody, thus removing theimmunoglobulins from the sample. The molecules that are not the desiredantibody were washed off and the bound antibody was eluted with 0.2 Mglycine, pH 2, then the pH was brought to neutral prior to dialysisovernight in PBS at 4° C., and the elutent containing the antibody wasused for the multi-site assay.

The detection antibody, MAb A4.6.1, binds rhVEGF165 with a Kd of 86 pM.Several lines of evidence suggest that this MAb binds rhVEGF near theKDR receptor binding region (Kim et al., supra (1992)).

The hybridoma cell line capable of producing MAb 3.5F8 was deposited atthe American Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va., 20110-2209, on Jul. 3, 2001, and has been assigned ATCCaccession number PTA-3499. The hybridoma cell line capable of producingMAb A4.6.1 was deposited at the ATCC (address above) on Mar. 29, 1991,and has been assigned ATCC accession number HB 10709. The deposits weremade under the provisions of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purpose of PatentProcedure and the Regulations thereunder (Budapest Treaty). This assuresmaintenance of a viable culture of the deposit for 30 years from thedate of deposit. The deposit will be made available by ATCC under theterms of the Budapest Treaty, and subject to an agreement betweenGenentech, Inc., and ATCC, which assures permanent and unrestrictedavailability of the progeny of the culture of the deposit to the publicupon issuance of the pertinent U.S. patent or upon laying open to thepublic of any U.S. or foreign patent application, whichever comes first,and assures availability of the progeny to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accordingto 35 USC §122 and the Commissioner's rules pursuant thereto (including37 CFR §1.14 with particular reference to 886 OG 638).

The assignee of the present application has agreed that if a culture ofthe material(s) on deposit should die or be lost or destroyed whencultivated under suitable conditions, the material(s) will be promptlyreplaced on notification with another of the same. Availability of thedeposited material(s) is not to be construed as a license to practicethe invention in contravention of the rights granted under the authorityof any government in accordance with its patent laws.

2.3. Biotinylation of MAb A4.6.1

The MAb A4.6.1 was biotinylated with biotinylamidocaproicacid-N-hydroxysuccinimide ester (Biotin-X-NHS) (Research Organics,Cleveland, Ohio) according to the following protocol. The MAb A4.6.1 wasdialyzed against 100 mM NaHCO_(3,) pH 8.5 overnight at 2-8° C. A totalof 60 μl of a 5-mg/ml solution of Biotin-X-NHS in DMSO was added to theMAb (adjusted after dialysis to a concentration of 2-10 mg/ml) using a1:10 (w/w) ratio of Biotin:MAb. This mixture was allowed to incubate fortwo hours at ambient temperature with gentle agitation, and the reactionwas stopped by the addition of 5 μl of ethanolamine. After conjugation,the antibody was extensively dialyzed against PBS at 2-8° C. with gentleagitation and PBS changed every two hours for a total of three times.

2.4. Multi-Site VEGF ELISA

Two MAbs, 3.5F8 (coat) and biotinylated A4.6.1 (detection), and one PAb(coat) as described above were used to develop a specific and sensitiveVEGF ELISA. In this ELISA, 100 μl/well each of MAb 3.5F8 and theaffinity-purified PAb were mixed together and then added to MaxiSorp™96-well microtiter plates (Nunc, Roskilde, Denmark) at 0.4 μg/ml each in0.05 M sodium carbonate, pH 9.6 . Following 24-74-hour incubation at2-8° C., the coated plates were washed 3 times with 400 μl ELISA washbuffer (PBS containing 0.05% TWEEN-20 ™ detergent) using a BIOTEK EL304™platewasher (Biotek Instruments, Winooski, Vt.), and blocked with ELISAblocking diluent at 200 μl/well (PBS containing 0.5% BSA, 0.05%TWEEN-20, ™ and 0.05% PROCLIN™ 300 antibiotic, pH 7.2) for 1-3 hours atambient temperature with agitation. After blocking, the plates werewashed again 3 times with 400 μl ELISA wash buffer. Then, 100 μl/well ofstandards, samples, or controls were added to duplicate wells andincubated for 1.5-2 hours at 37° C. with agitation. For quantitation ofrhVEGF165 in human plasma, the standard curve was prepared in ELISAdiluent (PBS containing 0.5% BSA, 0.05% TWEEN-20™, 0.05% PROCLIN™ 300, 5mM EDTA, and 0.35 M NaCl, pH 6.3±0.1). The standard curve was 128 pg/mldiluted serially 1:2 to 2 pg/ml. After the sample/standard incubation,the plates were washed six times with 400 μl ELISA wash buffer, and 100μl/well of MAb A4.6.1-Biotin, freshly diluted 1:200 to its optimalconcentration in ELISA diluent, was added to the plates. After a1.5-2-hour incubation at 37° C. with agitation, the plates were washedsix times as described above and 100 μl/well of strep-β-gal, diluted1:40 K in ELISA diluent, was added to the plates. After a 45-60-minuteincubation at 37° C. with agitation, the plates were washed 6 times asdescribed above and 100 μl/well of MUG/DMSO (1/100), freshly diluted to340 μg/ml in a solution of 0.1 M NaPO_(4,) containing 1 mM MgCl₂ at pH7.3±0.1, was added to the plates. The substrate reaction incubated for15-17 hours at 37° C. with agitation in the dark (plate was wrapped withfoil). The reaction was stopped by adding 150 μl/well of 0.15 M glycine,pH 10.5±0.1. The fluorescent unit (FSU) of the well contents was read ona CYTOFLUOR 4000™ fluorescent plate reader (PerSeptive Biosystems,Framingham, Mass.) using 360 nm excitation and 460 nm emission filters.A four-parameter curve fit program was used to generate a standardcurve, from which sample and control concentrations were interpolated.FSU readings were stable for at least 30 minutes at room temperatureafter 150 μL glycine was added.

2.5. Human Plasma Samples

The ability to accurately measure VEGF in human plasma was assessedusing several approaches. The effect of plasma on the assay sensitivityand performance was evaluated using rhVEGF165. Known amounts ofrhVEGF165 were added to individual human plasma samples and the percentrecovery determined as follows: (1) the amount of endogenous VEGF in thesample, determined from a parallel sample, was subtracted from the totalamount of VEGF measured in the sample, (2) the ‘recovered’ VEGF valuewas then divided by the amount of VEGF added to the sample andmultiplied by 100. The dilution linearity of rhVEGF165 added intoindividual human plasmas was also evaluated. In these studies, followingrhVEGF165 addition, each plasma sample was diluted 1:10 in ELISA diluentfollowed by serial 1:2 dilutions in ELISA diluent. High and low matrix(standard) controls were prepared in neat human EDTA plasma (frozen).They were diluted 1/10 in ELISA diluent for a final concentration of 10%plasma.

Endogenous VEGF levels were measured in individual human plasma samples.Blood from normal healthy individuals was drawn into 15% K3 EDTAVacutainer tubes (Becton Dickenson, San Jose, Calif.). The tubes werecentrifuged at 2000xg for 20 min and the plasma was collected. Plasmasamples were diluted 1:10 in ELISA diluent for use in the assay. Thedilution linearity of endogenous VEGF in selected samples was alsoevaluated as described above.

3. Results

3.1 Sample Stability

The stability of neat human EDTA plasma was examined for three freeze-and thaw cycles. The plasma received a dry ice treatment followed by agentle mixing in warm water in order to thaw. The data in Table 1 belowdemonstrate that there is no significant effect of quantitation of VEGFfollowing freeze- and thaw treatments. Therefore, human EDTA plasma isstable for three freeze- and thaw cycles.

TABLE 1 Net Freeze-Thaw Stability of Normal Human EDTA Plasma hu EDTAPlasma Two-Site Multi-Site 32 22.11 78.84 Fresh 25.4 75.16 1 freeze-thaw14.85 75.16 2 freeze-thaw 18.88 72.08 3 freeze-thaw mean 20.31 75.31stddev 4.51 2.77 % CV 2.2 4 33 35.44 100.24 Fresh 35.87 101.71 1freeze-thaw 35.44 101.71 2 freeze-thaw 39.19 101.71 3 freeze-thaw mean36.49 101.34 stddev 1.81 0.73 % CV 5 1 34 17.51 78.18 Fresh 25.03 94.6 1freeze-thaw 22.11 94.6 2 freeze-thaw 24.72 83.95 3 freeze-thaw mean22.34 87.83 stddev 3.48 8.16 % CV 16 9 39 29.35 84.62 Fresh 31.11 81.391 freeze-thaw 28.02 75.16 2 freeze-thaw 31.11 71.51 3 freeze-thaw mean29.90 78.17 stddev 1.50 5.93 % CV 5 8 38 23.43 83.95 Fresh 29.94 80.07 1freeze-thaw 21.8 83.95 2 freeze-thaw 24.72 66.84 3 freeze-thaw mean24.97 78.70 stddev 3.52 8.12 % CV 14 10 Mean 12 6 stddev = standarddeviation CV = coefficients of variation3.2 Limit of Detection

Approximately 20 replicates of the blank, 1, 2, 4 and 8 pg/ml standard,were assayed in the multi-site VEGF ELISA herein. The limit of detectionwas determined by the analyte (VEGF) concentration for which themeasured mean FSU response minus two standard deviations was greaterthan the mean FSU response plus two standard deviations of the blankfluorescence emission (460 nm). Results in Table 2 show that the limitof detection is 8 pg/ml in ELISA diluent. Since plasma samples aretypically diluted 1:10 to minimize matrix interference, as little as 80pg/ml, or 1.6 pM VEGF can be measured in the original sample.

TABLE 2 Limit of Detection (0.4 μg/ml, 0.4 μg/ml) std std std std std 0pg/ml 1 pg/ml 2 pg/ml 4 pg/ml 8 pg/ml replicates FSUs FSUs FSUs FSUsFSUs  1 1103 1277 1351 1546 2216  2 1091 1382 1359 1617 2292  3 11031336 1413 1745 2241  4 1180 1328 1986 1770 2266  5 1235 1321 1382 16542216  6 1135 1382 1631 1735 2216  7 1180 1306 1328 1692 2266  8 11541125 1512 1654 2241  9 1079 1351 1366 1682 2279 10 1129 1559 1529 16782384 11 1129 1314 1445 1654 2266 12 1263 1413 1299 1617 2228 13 10791382 1336 1631 2216 14 1235 1284 1851 1716 2565 15 1135 — 1711 1780 226616 1129 — 1590 2077 2266 17 1017 — 1445 1654 2279 18 1351 — 1445 17402371 19 1079 — 1546 1599 2318 20 2025 — 1626 1663 2228 mean 1192 13401508 1695 2281 std. dev. 211 94 183 108 82 +1 SD 1402 1434 1690 18032363 +2 SD 1613 1528 1873 1911 2445 −1 SD 981 1246 1325 1587 2199 −2 SD770 1152 1142 1479 21173.3 Testing and Preparation of Anti-VEGF PAb

Two different preparations of rabbit polyclonal antibody against rhVEGFpurified from the same rabbit but a different bleed were compared in theassay, using MAb 3.5F8 as the monoclonal antibody, and using 0.4 μg/mlof each type of antibody. The results, indicated in FIG. 1, show thatboth antibodies are suitable for use.

Rabbit polyclonal antibody elution was performed with glycine followedby guanidine and the resulting antibodies were used in the assay withthe preferred conditions herein. Results in FIG. 2 and Table 3 show thatthere is no significant difference between the two elution methods.However, the glycine elution seems to be slightly more sensitive.Comparison of normal human EDTA plasma samples as well as the High andLow Matrix controls show similar quantitation in both preparations. Theguanidine peak is more tightly bound to the VEGF than the glycine peak.

TABLE 3 Comparison of glycine and guanidine as eluents PAb (Glycine) +PAb (Guanidine) + Normal Human MAbs 3.5 F8/A4.6.1 MAbs 3.5F8/A4.6.1 %EDTA Plasma (pg/ml) (pg/ml) Recovery 1 37 48 77 2 41 34 123 3 112 90 1244 79 62 128 5 49 36 136 6 40 57 71 7 59 45 132 8 35 31 116 High Mat 99102 97 Low Mat 9 8 115 Mean % Recovery 1123.4 Robustness/Ruggedness

Inter-assay and intra-assay precision was evaluated for the low and highmatrix controls by ANOVA statistical analysis. Matrix controls wereprepared by spiking rhVEGF into neat human EDTA plasma at low and highconcentrations to fall within the assay range. Results show that theinter-assay variability (CV) ranges from 11-17% while the intra-assayvariability ranges from 8-14%. The data is summarized in Table 4.

TABLE 4 Reproducibility of the Matrix Controls Assay Name High (pg/ml)Low (pg/ml) kn324p2 107.5 6.8 kn324p2 111.2 11.6 kn324P3 93.8 10.4kn324p3 96.7 11.2 kn320p7 103.6 13.7 kn320p7 103.6 13.7 kn320p8 99.714.3 kn320p8 102.0 14.7 kn322p2 110.6 13.3 kn322p2 102.0 — kn322p3 97.014.4 kn322p3 103.9 13.9 kn322p1 99.0 12.0 kn322p1 95.2 12.3 kn320p3101.1 15.3 kn320p3 98.0 14.5 kn320p2 105.1 14.9 kn320p2 100.5 14.2kn320p1 106.0 14.1 kn320p1 103.4 13.9 kn319p2 87.1 10.2 kn319p2 100.29.3 kn319p1 97.6 9.4 kn319p1 99.6 9.1 kn316p1 92.4 11.7 kn316p1 97.911.3 kn313p1 117.8 18.8 kn313p1 111.4 16.2 kn212p2 92.4 14.6 kn212p292.4 15.7 kn311p1 123.9 14.2 kn212p1 103.8 13.9 kn212p1 93.2 13.7kn331p1 106.2 12.9 kn331p1 109.2 13.3 kn331p2 99.8 11.9 Matrix ControlsIntra-assay (% CV) Inter-assay (% CV) Low 14.0 11.0 High 8.0 17.03.5 Hook Effect

Several samples in the past have shown non-linearity of increasing VEGFmeasured with increasing sample dilution. Therefore, the multi-siteELISA herein was tested for a hook effect (side-by-side comparison withthe one- and two-site ELISAs). rhVEGF was diluted from 16 ng/ml to 1pg/ml in assay buffer. Results (depicted in FIG. 3) show that there isno significant drop in VEGF quantitation. However, a slight drop andplateauing effect can be seen from 512 pg/ml onward. Since the sampledilutions used in the multi-site ELISA assay herein give rise toconcentrations less than 128 pg/ml, the hook effect is not a concern.

3.6 Coat Maximization

The procedures for determining coat maximization were the same asdescribed above in the Methods section except that the concentration ofeither the PAb or the MAb 3.5F8 coat was varied. Specifically, for FIG.4 the concentration of MAb 3.5F8 was varied from 0.4 to 4 μg/ml whilekeeping constant the concentration of polyclonal antibody (at 1 μg/ml),and for FIG. 5 and Table 5 the concentration of polyclonal antibody wasvaried from 0.4 to 4 μg/ml while keeping constant the concentration ofMAb 3.5F8 (at 0.4 μg/ml).

While the 0.1 and 0.4 μg/ml concentrations of MAb 3.5F8 and of the PAbat the constant concentration of the other coat antibody wereessentially the same in VEGF quantitation for the low and high control,the upper limit of coat concentration (e.g., 0.4 μg/ml) is preferred tobetter the chances that the VEGF is captured. While the concentration of1 μg/ml of MAb 3.5F8 and PAb increased the amount of VEGF measured ineach case, such a concentration also gave higher background. Hence, theresults show that the preferred concentrations for both capture reagentsis about 0.4 μg/ml.

TABLE 5 Coat Maximization of the Polyclonal Antibody plus MAb 3.5F8 MAb3.5F8 0.4 μg/ml 0.4 μg/ml 0.4 μg/ml 0.4 μg/ml PAb 1 μg/ml 0.4 μg/ml 0.1μg/ml 0 High matrix 100.4 89.3 94.0 97.3 (pg/ml): Low matrix 22.4 9.710.1 5.4 (pg/ml): Eight separate 97.3 33.5 42.5 10.7 normal human 202.5106.5 77.0 19.7 plasma donors 162.3 52.9 59.0 26.8 (pg/ml): 92.0 27.529.8 11.1 115.6 41.4 42.5 11.0 202.5 70.8 69.7 15.8 409.9 196.6 207.994.9 512.1 25.7 275.0 113.03.7 pH Profile of the Multi-Site VEGF ELISA

A pH profile was performed to determine whether changing pH of the assaybuffer would increase or decrease recovery of VEGF in normal human EDTAplasma. Changing the pH could dissociate binding proteins or othercomplexes, if any, which would interfere with the MAb A4.6.1 detection.

The procedure for examining the pH profile of the assay was the same asdescribed in the Methods section above except that for the sampleincubation and biotin incubation, the assay buffer was adjusted usingNaOH or HCl, resulting in assay buffers ranging from pH 4 to 9. Astandard curve, a low and high matrix, and four normal human EDTA plasmasamples were evaluated from dilutions performed using these varying-pHassay buffers.

Results in FIG. 6 and Table 6 show that there was no recovery of VEGF atpH 4 and 5. However, pH 6-9 revealed good VEGF plasma recovery with theassay control within an acceptable range. There was no significantdifference in VEGF quantitation as a consequence of varying the pH ofthe assay buffer from 6 to 9. However, the preferred assay buffer is onewith a pH of about 6.35±0.1, which results in maximal VEGF binding andis appropriate for all dilution steps of the assay.

TABLE 6 VEGF Recovery Normal Human EDTA Plasma at varying pH pH 6 7 8 94 5 pg/ml pg/ml pg/ml pg/ml Normal Human EDTA Plasma 1 — — 198.9 122.0173.3 204.0 2 — — 141.4 81.7 138.7 138.6 3 — — 240.7 150.3 220.9 243.5 4— — 112.2 113.2 176.7 190.3 Controls High Matrix — — 104.5 105.0 93.8124.0 Low Matrix — — 25.7 25.1 19.7 16.93.8 Dilution Linearity

Approximately 85 pg/ml rhVEGF was spiked into neat human EDTA plasma andserially diluted to 1/10, 1/20, 1/40, and 1/80 and analyzed. Theresults, in Table 7 and FIG. 7, show the rhVEGF spiked in EDTA plasmashowed linear correlation to expected concentration, with a coefficientcorrelation of 0.996. The percent difference between dilution-correctedconcentration values determined for successive serial dilutions did notexceed a mean of 19%±7.5, as shown in Table 7.

TABLE 7 Dilution Linearity of Normal Human EDTA Plasma Normal Human EDTAPlasma [Measured] Corrected % (samples) pg/ml Dilution ConcentrationDifference S1 103 10 1026 — 62 20 1237 21 35 40 1416 14 20 80 1599 13 S2109 10 1088 — 59 20 1176  8 35 40 1416 20 22 80 1788 26 S3 104 10 1039 —60 20 1202 16 32 40 1278  6 21 80 1677 31 S4 88 10 878 — 52 20 1036 1832 40 1278 23 19 80 1528 20 S5 93 10 926 — 57 20 1136 23 32 40 1278 1218 80 1433 12 S6 119 10 1192 — 81 20 1612 35 47 40 1893 17 29 80 2298 213.9 Accuracy—Quantitation of VEGF in Human Plasma

Endogenous VEGF levels were measured in freshly-collected plasma fromseveral normal healthy individuals. The individual human EDTA plasmasamples were spiked with lowest, low, mid, and high concentrations ofrhVEGF so as to fall within the assay range of the standard curve.Endogenous VEGF concentrations were determined and subtracted form themeasure concentration to obtain comparison to the targeted spike.Results in Table 8 show that mean % recoveries were 99%, 113%, 106%, and118% for the high, mid, low, and lower spikes, respectively.

TABLE 8 Spike Recovery of rhVEGF in Human EDTA Plasma [Meas- [Measured-[Endogenous] ured] Endogenous] [Targeted] % pg/ml pg/ml pg/ml pg/mlRecovery High 18.6 100.5 81.9 85.3 96 Spike 22.5 114.8 92.3 85.3 10830.3 111.0 80.8 85.3 95 22.8 96.2 73.4 85.3 86 18.4 103.2 84.9 85.3 10034.1 121.1 87.0 85.3 102 Mean % Recovery 99 Mid 21.0 98.3 47.1 49.3 96Spike 9.1 89.1 80.1 49.3 162 10.8 66.8 55.9 49.3 113 21.0 59.5 38.5 49.378 9.1 66.8 57.8 49.3 117 Mean % Recovery 113 Low 4.5 32.9 28.4 22.9 124Spike 10.0 32.9 22.9 22.9 100 14.9 38.3 23.4 22.9 102 8.6 31.2 22.6 22.999 9.3 33.7 24.4 22.9 107 15.9 40.9 25.0 23.5 106 36.2 58.8 22.6 23.5 9638.9 66.4 27.5 23.5 117 Mean % Recovery 106 Lower 4.3 24.9 20.2 17.8 114Spike* 4.6 26.1 21.5 17.8 120 7.4 27.9 20.4 17.8 114 6.6 26.4 19.9 17.8111 10.8 33.1 22.2 17.8 124 5.5 29.6 24.0 17.8 135 3.9 22.7 18.6 17.8105 Mean % Recovery 118 *0.4 μg/ml MAb 3.5F8 + 0.4 □g/ml PAb coat3.10 Accuracy—Quantitation of VEGF in Normal Rat EDTA Plasma

An individual and two pooled male rat EDTA plasma samples were spikedwith low, mid and high concentration of rhVEGF so as to fall within theassay range of the standard curve. Endogenous VEGF concentrations weredetermined and subtracted from the measured concentration in order toobtain comparison to the targeted spike (dilution control). Spikes werethen diluted 1:2 in ELISA diluent to determine dilution linearity.Results in Table 9 show that mean percent recoveries range from 84-103%for the high, mid and low spikes that were greater than 6.25 pg/ml.

TABLE 9 VEGF Spike Recovery in Normal Rat EDTA Plasma [Expected][Measured] [Measured] [Measured] Dilution Male rat Male rat IndividualControl Pool % Pool % Male rat % Mean % pg/ml 1 pg/ml Recovery 2 pg/mlRecovery pg/ml Recovery Recovery High spike 151  160 106 155 103 148 98103 98 106 109 85 87 92 95 97 53 58 109 43 81 43 82 91 24 25 105 15 6118 76 81 Mid spike 44 41 94 40 92 39 89 92 22 27 125 20 93 16 71 96 1113 115 7 66 7 65 82  5 5 104 0 0 1 15 40 Low spike 20 18 88 19 93 12 6281 10 14 135 9 86 6 63 95  6 6 102 1 18 0 0 40  3 2 52 0 0 0 — 26 Lowerspike 10 10 98 8 76 5 49 74  5 8 149 3 47 2 42 79  4 8 202 0 0 0 0 67Endogenous LTS 43 393.11 Linearity of Normal Rat EDTA Plasma in ELISA Diluent

Rat EDTA plasma (2 male pools, 1 individual) was tested for linearity ofdilution. Neat plasma samples were spiked with low (20 pg/ml), mid (44pg/ml), and high (98 pg/ml) concentrations of rhVEGF and were seriallydiluted 1/10, 1/20, 1/40, 1/80 in ELISA diluent. Results in Table 10 andFIGS. 8A-8C show that normal rat plasma samples dilute linearlyfollowing a minimum 1/20 dilution in the assay range of 8-128 pg/ml.

TABLE 10 Summary of Linearity for Rat Plasma Samples (in units ofcoefficient of correlation (R)) Spike Rat pool 1 Rat pool 2 Rat 1 Mean Rvalue High 0.996 0.996 0.999 0.997 Mid 1 0.999 0.994 0.998 Low 0.9290.993 0.98 0.9673.12 Accuracy—Quantitation of VEGF in Normal Yorkshire Pig EDTA Plasma

Eight Yorkshire pig EDTA plasma samples (four males and four females)were spiked with low, mid, and high concentrations of rhVEGF so as tofall within the assay range of the standard curve. Endogenous VEGFconcentrations were determined and subtracted from the measuredconcentration in order to obtain comparison to the targeted spike(dilution control). Spikes were then diluted 1/10, 1/20, 1/40, 1/80 inELISA diluent to determine dilution linearity. Results, shown in FIG. 9A(females) and FIG. 9B (males), and in Table 11, show that normal ratplasma samples dilute linearly following a minimum 1/20 dilution in theassay range of 8-128 pg/ml.

TABLE 11 Summary of Linearity for Yorkshire Pig Samples GenderCoefficient of Correlation (R) Female 0.99718 Female 0.99917 Female0.99998 Female 0.99958 Male 1 Male 0.99965 Male 0.99995 Male 0.99961mean 0.99939 s.d. 0.00093491 % CV 0.0943.13 Detection of Various Forms of VEGF Using Three ELISAs

This experiment was designed to determine if the multi-site ELISA hereincould measure all the variants of VEGF. FIGS. 10A, 10B, and 10C show acomparison of the single-site, two-site, and multi-site ELISAs for VEGF,respectively. It can be seen by comparing these graphs that themulti-site assay herein is capable of capturing more VEGF variants.

3.14 Detection Using Two-Site, Multi-Site, or PAb as Coat

The multi-site ELISA herein using PAb and MAb 3.5F8 as coat antibodieswas compared to an ELISA using only MAb 3.5F8 or PAb as coat antibodyfor evaluating the amount of VEGF in normal human samples. The results,set forth in Table 12, show that the amount of VEGF detected in pg/mlwas much higher for the multi-site assay than for the assay with PAbalone or MAb 3.5F8 alone.

TABLE 12 Amount of VEGF in Normal Human Plasma Samples Using PAb Alone,MAb alone, or PAb and MAb Capture Reagent PAb to VEGF + MAb 3.5F8 PAb toVEGF MAb 3.5F8 NHP Sample # (Mean pg/ml) (Mean pg/ml) (Mean pg/ml) 1 49173 225 2 26 LTS 149 3 39 124 211 4 41 103 189 5 27 LTS 153 6 29 LTS 1497 16 LTS 159 8 25 LTS 144 9 21 LTS 122 10 36 148 185 11 24 72 171 12 23LTS 145 13 40 103 200 14 34 83 143 15 42 LTS 200 16 20 85 152 17 51 196285 18 25 LTS 145 19 20 LTS 154 20 20 LTS 143 21 23 LTS 155 22 28 77 16323 39 180 285 24 24 87 168 25 45 148 261 26 21 131 179 27 34 LTS 189 2818 LTS 131 29 50 159 251 30 73 LTS 359 31 21 85 149 32 42 214 237 33 33LTS 234 34 30 152 206 35 21 87 154 36 76 307 445 37 28 70 225 38 53 51304 39 32 84 193 40 28 LTS 106 41 44 105 275 42 32 44 217 43 28 69 19744 25 LTS 69 45 50 114 285 46 28 38 176 47 21 67 125 48 28 27 192 49 29LTS 159 50 18 27 107 LTS = not detectable3.15 Comparison of VEGF Levels in Normal Human Plasma and Normal HumanSerum Using Two-Site and Multi-Site ELISAs

Plasma and serum samples from normal human donors were analyzed by thetwo-site ELISA with MAb 3.5F8 as capture antibody and MAb A4.6.1 asdetection antibody and by the multi-site assay herein using the PAb andMAbs and procedures noted in the Methods. The results, summarized inFIGS. 11A and 11B for plasma and serum respectively, indicate that themulti-site assay herein detects more VEGF in both types of samples thanthe two-site assay.

3.16 Comparison of VEGF Levels in Normal and Cardiopathological PatientsUsing Single-Site, Two-Site, and Multi-Site ELISAs

Samples from normal human donors and from donors with cardiac diseasewho were enrolled in clinical trials sponsored by Genentech, Inc. toevaluate efficacy of TNK, a t-PA variant, were analyzed by thesingle-site ELISA with MAb 3.5F8 as coat and detection agent, by thetwo-site ELISA with MAb 3.5F8 as capture antibody and MAb A4.6.1 asdetection antibody, and by the multi-site assay herein using the PAb andMAbs and procedures noted in the Methods. FIG. 12 shows the amounts ofplasma VEGF in cardiac patients using all three assays, and FIG. 13 andTable 13 summarize the amounts of VEGF in normal and cardiac patientsusing the two-site and multi-site assays by the mean amount of VEGF,standard deviation, % CV and s.e.m. The results indicate that themulti-site assay herein detects more VEGF in both types of samples thanthe two-site assay.

TABLE 13 Sensitivity of Two-Site and Multi-Site Assays to VEGF in Normaland Cardiac Patients Normal Donors Cardiac Patients Two-site Multi-siteTwo-site Multi-site mean pg/ml 32.61 192.40 37.54 279.23 s.d. 13.0567.66 23.89 156.69 % CV 40.02 35.17 63.65 56.11 s.e.m. (Standard error1.84 9.56 5.34 35 mean)3.17 Comparison of Serum VEGF Levels in Lung Cancer Patients UsingTwo-Site and Multi-Site ELISAs

Serum samples from non-small cell lung carcinoma patients were analyzedby the two-site ELISA with MAb 3.5F8 as capture antibody and MAb A4.6.1as detection antibody and by the multi-site assay herein using the PAband MAbs and procedures noted in the Methods. The results, shown in FIG.14, indicate that the multi-site assay herein detects more VEGF in lungcancer samples than the two-site assay.

3.18 Levels of Serum VEGF in Diabetic Patients

Serum VEGF levels in normal humans and in patients with NIDDM (Type Idiabetes) and IDDM (Type II diabetes) were measured using the two-siteELISA (MAb 3.5F8 as coat and MAb A4.6.1 as detection agent) describedabove. FIG. 15 shows that the levels of serum VEGF in NIDDM and IDDMpatients were higher than in normal patients using this assay. Since themulti-site assay detects more VEGF than the two-site assay in otherdiseased patients, it would be expected that the multi-site assay hereinwould be suitable for detecting elevated levels of VEGF in diabeticpatients.

3.19 Specificity of PAb to VEGF Versus PAb to DNase in Multi-Site Assay

The two-site and multi-site ELISAs were carried out as described abovefor normal human plasma samples. In addition, a multi-site was carriedout using PAb to DNase rather than PAb to VEGF as coat reagent. All wereat the 0.4 □g/ml concentration. FIG. 16 and Table 14 show that the VEGFdetected by multi-site VEGF assay is specific. Results from the ELISAusing PAb to DNase plus MAb 3.5F8 show almost identical results as theELISA using MAb 3.5F8 alone as capture reagent, with a slope of 1.04(FIG. 16A).

TABLE 14 Human EDTA Plasma Evaluated for VEGF Amounts PAb to VEGF PAb toDNase (0.4 μg/ml) (0.4 μg/ml) Human EDTA MAb 3.5F8 and MAb 3.5F8 and MAb3.5F8 plasma (0.4 μg/ml) (0.4 μg/ml) (0.4 μg/ml) High matrix 147.9 147.4119.2 (pg/ml): Low matrix (pg/ml): 12.8 18.3 12.5 Seven separate 61.1164.0 59.5 normal human 33.8 120.4 48.5 plasma donors 34.6 174.3 64.5(pg/ml): 47.4 104.5 102.1 28.3 70.4 79.6 67.6 113.0 71.8 61.1 105.9 65.43.20 Summary of Preferred Assay and Results

TYPE OF ASSAY Immunoassay (Fluorimetric ELISA): mixture of a murineanti-VEGF monoclonal antibody (MAb 3.5F8) and a rabbit affinity-purifiedpolyclonal antibody for capture and an anti-VEGF monoclonal antibody(MAb A4.6.1) for detection. STANDARD rhVEGF Reference Material VEGF orequivalent. Standard curve will be diluted in ELISA diluent: PBS/0.5%BSA/0.05% Polysorbate20/0.05% PROCLIN ™ 300/5 mM EDTA/0.35M NaCl, pH6.35 ± 0.1 SPECIES Human, Rat, Yorkshire Pig QUALIFIED BIOLOGICAL Serum,EDTA Plasma, MATRIX ELISA diluent = Dilution buffer ASSAY RANGE 1-128pg/ml in ELISA diluent QUANTITATIVE 80 pg/ml to 1280 pg/ml for humanserum and EDTA RANGE IN plasma ( 1/10 minimum dilution) BIOLOGICAL 240pg/ml to 850 pg/ml for Yorkshire Pig EDTA Plasma MATRIX ( 1/20 minimumdilution) Minimum Endogenous values as low as 120 pg/ml will be reportedQuantifiable for information only with a % CV of approximately 60% inConcentration this range. Maximum 80 pg/ml to 1280 pg/ml for rat EDTAplasma Quantifiable Concentration INTRA-ASSAY Low 14% High 8% PRECISIONin Human EDTA Plasma INTER-ASSAY Low 11% High 8% PRECISION in Human EDTAPlasma ACCURACY 6 normal human EDTA plasma samples were spiked withhigh, mid and low concentrations of rhVEGF. Mean % recoveries were: LowMid High 106% 113% 99% 4 normal human serum samples were spiked with ahigh concentration of rhVEGF. Mean % recovery: High 113% 3 rat EDTAplasma samples were spiked with high, mid and low concentrations ofrhVEGF. Mean % recoveries in the quantitative range were: Low Mid High82% 91% 99% 4 female and 4 male Yorkshire pig EDTA Plasmas were spikedwith high, mid and low concentrations of rhVEGF. rhVEGF quantitationbelow 12 pg/ml (endogenous VEGF measured) will be reported forinformation only (mean % recovery below 12 pg/ml is approximately 60%).Mean % recoveries in the quantitative range were: Low Mid High n/a 86%107% SPECIFICITY rhVEGF, IGF, TNF NGF, hGH, IFN, llbllla, rhuMAb VEGF,anti-VEGF MAb 3.5F8 were spiked into human EDTA plasma. Only the rhVEGFspike had measurable values above the endogenous VEGF serum levels.LINEARITY AND rhVEGF was spiked into 6 different normal human EDTAINTERFERENCE plasma samples. Samples were serially diluted ½ to coverthe range of the standard curve. 4 dilutions were made from each sample.rhVEGF values obtained were plotted against 1/dilution and thecorrelation coefficient (R²) of a linear regression analysis wascalculated. Samples were linear across the range of the standard curve.n Mean R² SD 6 0.996 0.005 rhVEGF was spiked in 3 rat EDTA plasmasamples at high, mid and low concentration. Samples were linear acrossthe high and mid quantitative range. n Mean R² SD Spike 3 0.998 0.002High 3 0.998 0.003 Mid 3 0.967 0.034 Low rhVEGF was spiked into 8 EDTApig plasma samples and serially diluted ½ to cover the range of thestandard curve. 4 dilutions were made from each sample. Samples werelinear across the range of the standard curve. n Mean R² SD 8 1 0.001SAMPLE 5 normal EDTA plasma samples were tested for freeze-and-STABILITY thaw stability. Human EDTA plasma is stable for 3 freeze-and-thaw cycles. Mean % recoveries: 1X 101% 2X 101% 3X  93% RUGGEDNESS/pH profile indicates that the multi-site assay can tolerate ROBUSTNESSpH's ranging from 6-9. No quantitation of rhVEGF at pH 4 and 5. Noeffect on sample quantitation between a 1.5 and 16 hour sampleincubation. (% difference in control recover: H ctrl 19%, L ctrl 16%).Assay performed at room temperature. No effect on temperature incubation(1 hour vs. overnight at 37° C.) in VEGF quantitation of human plasmaand serum samples. Mean % recoveries: 1 hour overnight serum 113% 121%plasma 115% 113% ASSAY ACCEPTABILITY Matrix Controls WestgardMulti-rules: 2 SD from mean n (30% CV for Low Control) Buffer Controlsn/a Standard Curve: Correlation >0.994 Coefficient Precision (% CV) <10%Accuracy (% <20% differences) Regression <20% CV Parameters Samples <20%difference between dilutions. SPECIAL Samples containing rhuMAb VEGFwill interfere in the COMMENTS accurate quantitation of VEGF. Varioussamples were tested to improve sample linearity of endogenous VEGF(intending to release VEGF from possible binding proteins). Increasingamount of NaCl from 0.5-1.5 M in combination with varying pHs as well asthe addition of glycine or KSCN (pretreatment) or heat inactivation didnot improve dilution linearity or increase VEGF measured.4. Discussion

Little is known about the levels or the circulating forms of VEGF innormal individuals during growth, pregnancy, and old age or inpathophysiological disease states. Herein is described the developmentand characterization of a sensitive, high-throughput assay capable ofmeasuring various isoforms of VEGF and their levels in human plasma.This assay represents an important tool for measuring VEGF levels inboth normal individuals and in various disease states.

The multi-site VEGF ELISA herein can measure 165/165, 165/110, 121/121,and 110/110 VEGF variants equally well. With this assay, higher plasmaVEGF was detected in normal donors (192±68 pg/ml, n=50). For the same 18cardiovascular patients, a significantly higher plasma VEGF above normaldonors was detected (279±157 pg/ml, n=18, p<0.001). See FIG. 13. Indeed,monoclonal antibody MAb 3.5F8 plus affinity-purified polyclonal antibodyagainst an irrelevant protein did not generate any additional signalabove that of MAb 3.5F8 alone. It is concluded that besides intact VEGF,other VEGF variants and isoforms are present in the circulation of bothnormal donors and cardiovascular patients. Ability to demonstrate thatthe receptor binding domain of VEGF is accessible for binding may be animportant feature for any assay intended to understand the biologicalactivity of VEGF in the circulation. The fluorometric substrate,strep-β-gal/MUG, is preferred for use in the detection system so thatthe ELISA can detect endogenous VEGF levels in normal individuals. Theuse of this substrate and the determination of the best ELISA diluentresulted in much lower background absorbance, which was preferred toachieve the increase in the assay sensitivity.

The multi-site ELISA described herein is highly specific due to thechoice of antibodies used for capture and detection. One of the coatantibodies, MAb 3.5F8, binds near the heparin binding region of VEGF(residues 111-165) and the other coat antibody, the rabbit polyclonalantibody binds VEGF. The detection antibody, MAb A4.6.1, binds in theKDR receptor binding region (residues 1-110) of the molecule, yielding aspecific ELISA for VEGF.

The specificity of this multi-site ELISA will be important as thebiology of VEGF is better understood. Keyt et al., supra (p. 7788) havedemonstrated that the different VEGF variants examined in this studyhave varying bioactivities in vitro. Knowledge of assay specificity willalso be extremely important in evaluating clinical data and comparingdata between laboratories.

Published reports (Kondo et al., supra (1994); Takano et al., supra(1996); Rodriguez et al., supra) have noted that serum VEGF levels wereelevated in cancer patients. Considering that angiogenesis is a generalphenomenon in solid tumor progression, and that expression of VEGF, atumor angiogenesis factor, is observed in a wide variety of tumor cellsof various origins, measurement of circulating VEGF levels has potentialas a non-invasive diagnostic marker for a wide spectrum of solid tumors.

In conclusion, a sensitive ELISA that measures most molecular forms ofVEGF has been developed. In accordance with the present invention,antibodies are raised in animals against human VEGF, with the C-terminalspecific antibody being a monoclonal antibody and thewhole-VEGF-specific antibody being a polyclonal antibody, preferablyaffinity purified. These two antibodies are used as coat antibodies(immobilized capture reagents) on a solid support such as microtiterplates. The antibody used for detection can be either polyclonalantibodies or monoclonal antibodies provided they are specific for theKDR and FLT1 binding domain regions of human VEGF.

Accurate and sensitive ELISAs like the one described herein are deemedimportant in helping to understand VEGF levels in various diseasestates. A better understanding of both VEGF levels and the dominantisoforms present in both normal individuals and in pathophysiologicaldisease states will enhance knowledge of the role of VEGF in normal andpathologic angiogenesis.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

1. A method for detecting multiple isoforms of vascular endothelialgrowth factor (VEGF) in a biological sample, comprising: (a) incubatinga biological sample with a capture reagent immobilized on a solidsupport to bind multiple isoforms of VEGF to the capture reagent at a pHof about 6.0 to about 9.5, wherein the capture reagent comprises amixture comprising a polyclonal antibody that binds VEGF and amonoclonal antibody that specifically binds to amino acid residues111-165 of human VEGF; and (b) detecting VEGF bound to the immobilizedcapture reagent by contacting the bound VEGF with a detectable antibodythat binds to N-terminal amino acid residues 1-110 of VEGF.
 2. Themethod of claim 1, wherein the pH is about 6.0 to about 7.0.
 3. Themethod of claim 2, wherein the pH is about 6.0 to about 6.5.
 4. Themethod of claim 3, wherein the pH is about 6.35.
 5. The method of claim1, wherein the incubation of step (a) is at a temperature of about 0° C.to about 40° C.
 6. The method of claim 5, wherein the incubation is at atemperature of about 36° C. to about 38° C.
 7. The method of claim 1,wherein the incubation of step (a) is about 0.5 to about 3.0 hours. 8.The method of claim 7, wherein the incubation is about 1.5 to about 3.0hours.
 9. The method of claim 1, wherein the incubation of step (a) isnot greater than about 10 hours.